Sclerostin and the inhibition of Wnt signaling and bone formation

ABSTRACT

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass (HBM). In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293 cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by over-expression of Wnt coreceptor LRP5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transducing canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at the late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the HBM phenotype associated with the loss of sclerostin may at least in part be attributed to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.

REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of patent application Ser. No.11/084,668, filed on Mar. 18, 2005, which is a continuation-in-part ofpatent application Ser. No. 10/849,067, filed on May 19, 2004, whichclaims priority to U.S. Provisional Application No. 60/504,860, filed onSep. 22, 2003.

FIELD OF THE INVENTION

The present invention relates to the protein sclerostin, an antagonistand/or inhibitor of Wnt proteins. Sclerostin inhibits Wnt signaling andthus the formation of bone when it binds to the LRP5 receptor or theLRP6 receptor (LRP5/6). The invention relates to the field oftherapeutic methods, compositions and uses thereof, in the treatment ofbone fractures, bone disease, bone injury, bone abnormality, tumors, orgrowths. More particularly, the compositions and methods are directed tocompounds that block sclerostin, thereby allowing bone formation tooccur. The compounds were identified from a National Cancer Institute(NCI) database through various screening methods and assays. Thesecompounds could also be modified to create derivatives or analogues notfound in the NCI database or in nature which also function effectively.

All patents, patent applications, patent publications, scientificarticles, and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the invention pertains.

BACKGROUND OF THE INVENTION

The Wnt family of secretory glycoproteins is one of the major familiesof developmentally important signaling molecules which play importantroles in embryonic induction, generation of cell polarity, andspecification of cell fate. Both genetic and biochemical studiesindicate that frizzled (Fz) and LRP5/6 are co-receptors for transducingcanonical Wnt signaling that eventually leads to the stabilization ofβ-catenin and regulation of gene transcription through transcriptionregulators including lymphoid enhancing factor-1 (LEF-1) and T cellfactors (TCF). Wnt signaling is also regulated by a number of naturallyoccurring antagonists that include Dickkopf (Dkk) molecules. The firstDkk (Xenopus Dkk-1), was initially discovered as a Wnt antagonist thatplays an important role in head formation. To date, four members of Dkkhave been identified in mammals. However, only the first two members(Dkk1 and Dkk2) have been well documented to function as antagonists ofcanonical Wnt signaling. Both Dkk1 and Dkk2 antagonize canonical Wntsignaling by simultaneously binding to LRP5/6 and a single transmembraneprotein called Kremen. It has been further demonstrated that the second,but not the first, Cys-rich domains of Dkk1 and Dkk2 inhibit canonicalWnt signaling.

A myriad of evidence demonstrates that an increase in LRP5/6-mediatedcanonical Wnt signaling leads to an increase in bone mass.Loss-of-function mutations in LRP5 are responsible for humanosteoporosis-pseudoglioma syndrome (OPPG), an autosomal recessivedisorder, while putative gain of function mutations, including theGly171 to Val substitution, are associated with human high bone mass(HBM) phenotypes. In addition, mice in which the LRP5 gene wasinactivated by gene targeting showed phenotypes similar to those of OPPGpatients, and the transgenic expression of LRP5_(G)171V in mice resultedin HBM. Moreover, mouse primary osteoblasts showed reducedresponsiveness to Wnt and low proliferation indices in the absence ofLRP5, and canonical Wnts or activated β-catenin stimulated the canonicalWnt signaling activity and induced the production of an osteoblastmarker alkaline phosphatase (AP) in osteoblast-like cells. The findingthat inactivation of the Wnt antagonist sFRP1 enhances trabecular boneaccrual further supports the idea that canonical Wnt signaling enhancesbone formation. Dkk1 is expressed in differentiated osteoblast cells andosteocytes and the G171V mutation in LRP5 may cause the HBM phenotype byattenuating the antagonistic effect of Dkk1 on canonical Wnt signaling.

Itasaki et al. described a new Wnt antagonist called WISE. WISE appearsto be a context-dependent regulator of Wnt signaling; it may inhibit orstimulate Wnt signaling in different assays in Xenopus. WISE was alsoshown to bind to LRP6 and compete with Wnt8 for binding to LRP6. WISEshares 38% amino acid identity with sclerostin, the gene product ofSOST. Loss of function mutations of SOST are responsible for anautosomal recessive sclerostin skeletal disorder. Previous studies haveshown that sclerostin was highly expressed in osteocytes and that itmight act as a bone morphogenetic protein (BMP) antagonist, but anotherstudy suggested that sclerostin might not be a functional BMP antagonistand speculated that it might modulate Wnt signaling. In this report, wenow clearly demonstrate that sclerostin can bind to both LRP5 and LRP6and act as a Wnt antagonist. Because sclerostin expression occurs afterpeak Wnt7b expression during the osteogenic differentiation, thereduction in sclerostin-mediated antagonism of Wnt signaling contributesto the increases in bone mass associated with SOST.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions thataddress several problems related to bone remodeling, such asosteoporosis and other bone diseases. The invention also provides forthe use of compositions to aid in the healing of fractures or otherinjuries or abnormalities of bone. In particular, the invention providesa process for promoting bone formation in a mammalian subject comprisingadministering to the subject an effective amount of compounds whichprevent the binding of sclerostin.

The invention further provides for gene therapy methodologies forclinical conditions characterized by insufficient bone formationcomprising administering an effective amount of a compound that preventssclerostin binding, or by causing a decrease in the expression ofsclerostin.

In other aspects of the invention, gene expression, detection andquantification of sclerostin or related proteins serve as potentialdiagnostic methods for a variety of bone diseases.

The present invention is also directed to methods and compositions thataddress tumors or other bone growths.

The present invention has identified compounds which, when provided to acell, bind to, interact with, or fit into sites or cavities found on thedomains of the co-receptors involved in the stimulation, enhancement,inhibition or regulation of bone formation, or bone remodeling. Thesereceptors include the LRP5 receptor, the LRP6 receptor, the frizzledreceptor or any other receptor involved in the LRP5 or LRP6 (LRP5/6)receptor system.

The compounds were identified using screening methods described inpatent application Ser. No. 10/849,067. These compounds were found todisrupt the sclerostin and LRP5/6 interaction. Other compounds inhibitedWnt signaling by inhibiting the binding of Wnt to LRP5/6. The compoundsof the present invention are non-native, or exogenous compounds whichare not present in the cell, but originate from an outside source.Specifically, the compounds identified as IIIC3 (NCI₈₆₄₂) and IIC8(NCI₃₆₆₂₁₈) were found to disrupt the sclerostin and LRP5/6 interaction.As shown on FIG. 5, the binding of sclerostin-AP (a fusion protein ofsclerostin and alkaline phosphatase) to LRP5 decreased when either 111C3or 11C8 was added. The compounds bind to LRP5/6, and therefore preventsclerostin from binding, blocking Wnt and probably inhibiting boneformation.

DETAILED DESCRIPTION OF THE INVENTION

Because of the homology shared between WISE and sclerostin, experimentswere carried out to determine whether sclerostin would exert an effecton canonical Wnt signaling. The effect of conditioned medium (CM)containing mouse sclerostin on Wnt3a-induced activation of canonical Wntsignaling was determined using the LEF-1-based reporter gene assay inhuman embryonic kidney (HEK) cells. Sclerostin-containing CM showedmarked inhibition of Wnt3a activity in a dose-dependent manner (FIG.1A). Because control CM started to show significant inhibition at 50micro-liters, higher doses were not tested. To further confirm thiseffect of sclerostin, sclerostin and another canonical Wnt, Wnt1, werecoexpressed in HEK cells, and sclerostin showed up to 60% inhibition ofthe activity of coexpressed Wnt-1 (FIG. 1B, bars 2&4). Interestingly,coexpression of LRP5 abolished the antagonistic effect of sclerostin onWnt signaling, and a slight stimulation of Wnt1 signaling by sclerostinwas even observed in the presence of coexpressed LRP5 (FIG. 1B, bars 6 &8). The effect of sclerostin on Wnt signaling in an osteoblastic cellline MC3T3 was also examined. Expression of sclerostin also showed up to70% inhibition of Wnt-1 activated reporter gene activity in MC3T3 cells(FIG. 1C, bars 2&4). Once again, expression of LRP5 reversed theinhibition (FIG. 1C, bars 6 & 8). However, there was no increase in Wnt1activity in MC3T3 cells when sclerostin and LRP5 were expressed with Wnt(FIG. 1C). Nevertheless, all these results clearly demonstrate thatsclerostin antagonizes canonical Wnt activity activated by canonicalWnts when LRP5 is expressed at endogenous levels.

To understand how sclerostin antagonizes canonical signaling,experiments were carried out to determine if sclerostin binds to LRP5/6directly. The binding of sclerostin-alkaline phosphatase (AP) fusionprotein to cells expressing exogenous LRP5 or LRP6 were measured, withthe same methods used for Dkk1-AP. As shown in FIG. 2A, sclerostin-APshowed a LRP6-binding curve similar to Dkk1-AP, suggesting thatsclerostin-AP has an affinity for LRP6 comparable to that of Dkk1-AP,which was previously determined to be sub-nanomolar. The binding ofsclerostin-AP and Dkk1-AP to LRP5-expressing cells revealed thatsclerostin. -AP and Dkk1-AP also have similar affinities for LRP5 (FIG.2B). To delineate which regions of LRP5 are responsible for the bindingof sclerostin-AP, we measured the binding of sclerostin-AP to two LRP5mutants that lack either the first or last two YWTD-EGF repeat domains.These mutants are designated as LRP5R12 or LRP5R34, respectively (FIG.2E). While Dkk1-AP was capable of binding to both LRP5 mutants (FIG.2D), sclerostin-AP could only bind to LRP5R12, but not LRP5R34 (FIG.2C).

We have previously shown that LRP5R12 was still able to transduce Wntsignaling, suggesting that this LRP5 mutant may still retain theWnt-binding sequences. To determine if sclerostin and Wnt compete witheach other for the binding to LRP5R12, the binding of sclerostin-AP tocells expressing LRP5R12 in the presence or absence of Wnt3a CM wasmeasured. The presence of Wnt3a did not affect the binding ofsclerostin-AP to LRP5R12 at all (FIG. 3A). In contrast, the presence ofDkk1 completely blocked the binding of sclerostin-AP to LRP5R12 (FIG.3B). In an attempt to further delineate sclerostin binding sequences onLRP5, two additional LRP5 mutants were constructed, which lack thesecond to fourth YWTD-EGF repeat domains and the first, third, andfourth YWTD-EGF repeat domains, respectively. However, these two LRP5mutants did not bind to either sclerostin-AP or Dkk1-AP, nor did theytransduce Wnt signaling. These results suggest that, either both firstand second YWTD-EGF repeat domains are required for the binding ofsclerostin to LRP5 or these LRP5 mutants were incorrectly folded.

Several LRP5 mutations in the first YWTD-EGF repeat domain have beenfound to be associated with HBM. We have previously characterized one ofthe mutations, G171V, and found that this mutation interfered with theinteraction of LRP5 with its chaperon Mesd, resulting in poortransportation of LRP5 to cell surfaces. Because this LRP5 mutant wasstill able to transduce signals intracellularly for autocrine Wnts, itwas thought that the mutation may increase Wnt signaling by retainingthe LRP5 receptor inside the cells from extracellular antagonists suchas Dkk1 because Dkk1 is highly expressed in osteocytes. The finding ofsclerostin as a new Wnt antagonist, which is known to be expressed inthe bone and osteocytes, may provide alternative explanations for theeffects of the G171V mutation, which is located in the first YWTD-EGFrepeat domain and within the sclerostin-binding region. One of suchexplanations may be that the G171V mutation directly interferes with thebinding of LRP5 to sclerostin. To test this possibility, we measured andcompared the binding of sclerostin-AP to LRP5G171V with that of Dkk1-AP.As we have previously shown, cells expressing LRP5GV have a five-foldlower apparent binding to Dkk1-AP than cells expressing wildtype LRP5(FIG. 3C) due to the interference of the chaperon's function by themutation. Similarly, cells expressing LRP5GV also showed a reduction inthe binding of sclerostin-AP to the same degree (FIG. 3B). As the G171Vmutation does not directly interfere with the interaction between LRP5and Dkk1, it is also unlikely that the mutation interferes with theinteraction between LRP5 and sclerostin. The observation that LRP5GVcould still reverse sclerostin-mediated inhibition of Wnt activity inthe same dose range as the wildtype LRP5 (FIG. 1B,C) provides furthersupport for the idea that the G171V mutation does not interfere with theinteraction between LRP5, or LRP6 and Dkk1.

Sclerostin has been previously shown to be primarily expressed inosteocytes. We examined sclerostin expression in relation to Wnt7Bexpression during primary calvarial osteoblast differentiation. Wepreviously identified Wnt7b, a canonical Wnt that can stabilizeβ-catenin, as the only Wnt that showed drastic changes in its expressionlevels during primary bone marrow osteoblast differentiation. Similarly,the expression levels of Wnt7b showed drastic changes during calvarialosteoblast differentiation; the expression of Wnt 7b peaks at Day 8 andthen receded to lower levels, preceding the expression of osteogenicmarker osteocalcin and another Wnt antagonist Dkk1 (FIG. 4A). In situhybridization further confirms the conclusion on Wnt 7b expression inthat Wnt7b mRNA was detected primarily in early undifferentiatedosteoblasts in a mouse long bone (FIG. 4B). The expression of sclerostinshowed a similar time course to that of osteocalcin and only occurred atthe late stages of the differentiation when presumably osteocytes areforming in the mineralized matrix (FIG. 4A). This pattern of sclerostinexpression is consistent with previous in vivo observations thatsclerostin is expressed in osteocytes buried in the bone matrix and mayplay a role in mechanical loading. On the basis of the expressionpatterns of sclerostin and Wnt7b, we postulate that sclerostincontributes to the G171V-associated HBM phenotype even though sclerostinmay not directly interfere with Wnt binding or the mutation does notaffect sclerostin binding to LRP5. As suggested by our hypothesis thatthe G171V mutation may hide the receptor from paracrine antagonistswithout diminishing the signaling ability of the mutant receptor forautocrine Wnt, sclerostin, which is only produced by well differentiatedosteoblasts or osteocytes, would be one of such paracrine antagoniststhat conceivably has less access to LRP5G171V than the wildtype LRP5.Thus, the G171V mutation may increase Wnt activity by attenuating theantagonism of canonical Wnt signaling by not only Dkk1, but alsosclerostin and potentially other paracrine Wnt antagonists present inthe bone.

In previous studies, sclerostin was shown to act as a BMP antagonist. Itis convincing that sclerostin has a reasonably high affinity for BMP6and BMP7. However, the biological effects of sclerostin on BMP wasmerely determined by measuring BMP-induced alkaline phosphtase (AP)activity 3-6 days post ligand addition in osteoblastic cells. This APactivity readout is not specific for BMP activity. In fact, canonicalWnts can also stimulate AP activity in these types of cells. Incontrast, our Wnt reporter gene assay is specific for canonical Wnt andcannot be activated by BMP in HEK cells (data not shown). In addition,in the assay using CM, we measured the effect of sclerostin in 6 hours(FIG. 1A). Given the recent observations that sclerostin failed toinhibit early responses elicited by BMP, we believe that it is morelikely that sclerostin is biologically a canonical Wnt antagonist andthat its effects on bone mass is probably primarily attributed to itsantagonistic effect on canonical Wnt signaling.

As shown in FIG. 2, sclerostin binds to the first two YWTD-EGF repeatdomains of LRP5, which are also required for transducing Wnt signals.However, our evidence suggests that the antagonistic effect ofsclerostin is unlikely due to direct competition with Wnt for LRPbinding, because 1) Wnt3a failed to inhibit the binding of sclerostin-APto LRP5; and 2) LRP5 could reverse the inhibitory effect of sclerostinon canonical Wnt signaling. The latter observation is reminiscent of theeffect of Dkk1 on Wnt signaling as Dkk1 suppression of Wnt signaling canalso be reversed by exogenous expression of LRP5/6. The reason for theability of LRP5/6 molecules to reverse Dkk's effects is becauseDkk-mediated antagonism requires another protein Kremen. When Kremen iscoexpressed with LRP5/6, Dkk-mediated inhibition could be restored.Although Kremen had no effect on sclerostin-mediated antagonism, wesuspect that a similar mechanism may be used by sclerostin to inhibitWnt signaling. In other words, there may be accessory proteins likeKremen that may be required for sclerostin to function efficiently as anantagonist. Recently, noggin has been shown to directly interact withsclerostin and inhibit noggin's capacity to inhibit BMP signaling. Thus,noggin, once bound to sclerostin, might inhibit sclerostin capacity tomodulate Wnt signaling. In addition, the observation that sclerostinshowed slight stimulation of the LEF-1 reporter gene activity in thepresence of exogenous LRP5 or LRP5GV suggests that sclerostin may be apartial agonist under certain circumstances, even in mammalian systems.

The present invention provides methods for promoting or regulating boneformation or bone remodeling comprising administering at least onenon-native compound, a fragment of a non-native compound, or anycombination thereof. A non-native compound is defined as a compound thatis not naturally found in a mammalian subject, a human body inparticular. A non-native compound may also comprise an artificiallymanufactured compound that is identical to a compound that is naturallyfound in the human body. When the non-native compound or compounds bindto a receptor or co-receptor involved in bone formation or boneremodeling, the binding of sclerostin is prevented, thereby allowingbone to form.

Two or more non-native compounds may join together directly throughcross-linking, for example, or indirectly through a linker arm. Each ofthese linked compounds may dock in different locations on the samebinding site, protein or receptor. Each of these linked compounds mayalso dock in different locations on different binding sites, proteins orreceptors.

The compounds or fragments of compounds may be a small molecule,protein, peptide, polypeptide, cyclic molecule, heterocyclic organicmolecule, nucleic acid, lipid, charged lipid, polar lipid, non-polarlipid, sugar, glycoprotein, glycolipid, lipoprotein or chemical. Thecompounds or fragments may also be agonists, antagonists, partialagonists, or any combination of the aforesaid.

The compound may be administered by inhalation, orally, intravenously,intraperitoneally, intramuscularly, parenterally, transdermally,intravaginally, intranasally, mucosally, sublingually, topically,rectally or subcutaneously.

The present invention also provides a method for identifying a compoundor drug candidate that will bind to a signal peptide or protein involvedin protein-protein interactions, to inhibit or promote the occurrence ofsubsequent events. Specifically, the compound or drug candidate willbind to the receptor protein to inhibit or promote bone formation orbone remodeling. The first step involves determining the virtual orcomputational structure of the receptor protein through the use ofvarious methods such as amino acid sequencing, X-ray crystallography,NMR, analogs or derivatives of the receptor protein, or any combinationof the aforesaid methods. In a preferred embodiment, the protein isnon-soluble or membrane-bound.

The next step involves identifying a particular binding cavity site ordomain on the receptor protein through the use of experiments based onbiological function comprising mutational analysis, chemicalmodifications (of amino acids, for example), co-crystallography, NMR orany combination of the aforesaid methods. Using the results obtainedfrom these experiments, such as mutations and chemical modifications, aspecific binding site or domain is identified within the binding cavityto which the compound or drug candidate will bind. The entire bindingcavity or a specific binding site within the cavity may be used toscreen for a compound that fits and binds. The screening is conductedusing the UNITY™ program. The docking of the compound into the cavity iscarried out through the use of the Flexx™ program. The compound with thehighest binding affinity or the lowest binding energy using the Cscore™program is then selected. The ultimate goal is to select a compound ordrug candidate with the best fit.

A preferred embodiment of the invention is a method for preventing orblocking bone formation in a mammalian subject by administeringsclerostin.

Another preferred embodiment of the present invention is a method forthe treatment of abnormal bone growth comprising administering anantibody for sclerostin, or any other compound or fragment of a compoundwhich decreases or eliminates sclerostin, or decreases or eliminates theaffinity of sclerostin to a receptor or co-receptor involved in boneformation or bone remodeling.

Materials and Methods Cell Culture, Transfection, Preparation of CM, andLuciferase Assay.

Human embryonic kidney cell (HEK) line A293T and mouse osteoblastic cellline MC3T3 were maintained and transfected as previously described. Forluciferase assays, cells in 24-well plates were seeded at 5×10⁴cells/well and transfected with 0.5 μg DNA/well using Lipofectamine Plus(Invitrogen, CA), as suggested by the manufacturer. The LacZ plasmid wasusually used to make DNA concentrations equal for each transfection.Cell extracts were collected 24 hr after transfection. Luciferase assayswere performed as previously described. Luminescence intensity wasnormalized against fluorescence intensity of GFP. For preparation ofDKK1-AP and sclerostin-AP containing CM, HEK cells were seeded in 6well-plates at 4×10⁵ cells/well and transfected with 1 μg DNA/well. CMswere collected 48 hr after transfection.

Construction of Expression Plasmids and Mutagenesis.

The wild-type and mutant forms of human LRP5, LRP6, mouse Wnt1, DKK1,sclerostin, and DKK-2 were generated by PCR using the high fidelitythermostable DNA polymerase Pfu Ultra (Stratagene, Calif.). nucleotidesequences were verified by DNA sequencing. HA or Flag epitope tags wereintroduced to the C-termini of the full-length and mutant molecules. Theexpression of these molecules was driven by a CMV promoter. The LEF-1reporter gene constructs were kindly provided by Dr. Grosschedl.

DKK1-AP and Sclerostin-AP Binding Assay.

HEK cells in 24-well plates were transfected with LRP5 and its mutants.One day later, cells were washed with cold washing buffer (HBBScontaining BSA and NaN₃) and incubated with mouse DKK1-AP orsclerostin-AP CM on ice for two hours. Then, cells were washed threetimes with the washing buffer and lysed. The lysates were heated at 65°C. for 10 min, and its AP activity was determined using a Tropixluminescence AP assay kit. The immunoprecipitation assays were carriedout essentially as previously described.

Primary Calvarial Osteoblast Culture.

Mouse calvarial osteoblast cultures from 5 day old mice were generatedas previously described and were induced to undergo osteogenicdifferentiation in the presence of 8 mM β-Glycerophosphate, and 50 ug/mlascorbic acid. Media were changed every two days.

Quantitative PCR Analysis.

Total RNA was isolated using the TRIzol reagent (Invitrogen) accordingto manufacturer's instructions. For QPCR analysis, RNA wasreverse-transcripted by SuperScript™ First-Strand Synthesis System forRT-PCR (Invitrogen). QPCR was carried out using QuantiTect™ SYBR GreenPCR kit (Qiagen) on a DNA Engine OPTICON™ (MJ Research Inc.) instrument.B-actin was used as an internal reference for each sample. Using aformula previously described, the relative change in mRNA levels wasnormalized against the β-actin mRNA levels.

In Situ Hybridization.

The full-length coding region of Wnt7b was used to synthesize anti-senseand sense probes. The probes were labeled with Digoxigenin using an RNALabeling Kit (Roche, Indianapolis, Ind., USA). Sections of the tibiafrom a 3-weeks old mouse were dewaxed, rehydrated and fixed again with4% paraformaldehyde. Then the section s were treated with 2% glycine andProteinase-K and acetylated using an acetic anhydride/TEA solution,followed by hybridization with a digoxygenin-labelled probe. Afterwashing the sections with 50% formamide, 5×SSC, 5% SDS for 30 minutes at70° C. twice and 50% formamide, 2×SSC for 30 minutes at 65° C., thesections were incubated with anti-digoxigenin-alkaline phosphataseantibody followed by Nitro Blue etrazolium/4-bromo-5-chloroindolylphosphate, which yields a purple blue color. The sections werealso counterstained with methyl green (nuclei) and orange G(cytoplasma).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sclerostin antagonizes canonical Wnt signaling.

A) Effects of sclerostin CM on Wnt3a CM. Wnt3a CM (25 ul) were mixedwith varying amounts of Sclerostin CM (SCM) or control CM (CCM) andadded to HEK cells transfected with the LEF-1 reporter gene. Six hourslater, cells were lysed, and luciferase activity was determined. Theactivity in the absence of SCM is taken as 100%. Wnt3a CM increasedreporter gene activity by 5 folds. Expression of Flag tagged sclerostinwas detected by an anti-Flag antibody (insert). B,C) Effects ofcoexpressed sclerostin on Wnt1 signaling in HEK (B) and MC3T3 (C) cells.Cells were transfected with cDNAs encoding Wnt1, Sclerostin (Scl),wildtype LRP5 (Wt), or G171V LRP5 (GV) as indicated in the figure andthe LEF-1 reporter gene and a GFP expression plasmid. One day later,cells were lysed, and the GFP levels and luciferase activities weredetermined and normalized against GFP levels.

FIG. 2. Binding of sclerostin-AP to LRP5 and its mutants.

A,B) Binding of Dkk1-AP and sclerostin-AP to full length LRP6, LRP5 orLacZ. HEK cells were transfected with the full-length LRP6 (A) or LRP5(B). Binding of Dkk1-AP or sclerostin-AP (Scl) was determined asdescribed in the Method. Binding to cells transfected with controlplasmid LacZ was subtracted as non-specific binding. Specific binding ispresented in the charts. B,C) Binding of Dkk1-AP and sclerostin-AP toLRP5 mutants. HEK cells were transfected with LacZ or LRP5 mutants asindicated. Binding of sclerostin-AP (C) and Dkk1-AP (D) was determined.(E) Schematic representation of LRP5 mutants.

FIG. 3. Effects of Wnt3a, Dkk1, and LRP5 mutation on sclerostin binding.

A) HEK cells were transfected with LRP5. Binding of sclerostin-AP (5 ul)was determined in the presence of control CM (CCM) or Wnt3a CM (WCM, 100ul). B) HEK cells were transfected with LRP5R12. Binding ofsclerostin-AP (50 ul) was determined in the presence of buffer orrecombinant Dkk1 (10 nM). C) HEK cells were transfected with LRP5 (blackbars) or LRP5G171V (white bars). Binding of Dkk1-AP (Dkk) orSclerostin-AP (Scl) was determined. In all these binding assays, bindingto cells transfected with control plasmid LacZ was subtracted asnon-specific binding. Specific binding is presented in the charts.

FIG. 4. Expression of Wnt7b, sclerostin, Dkk1, and osteocalcin.

A) Primary calvarial osteoblast cultures were established from 5 daysold mice. Differentiation inducers were added on day 5. Relativeexpression levels of Wnt7b, sclerostin (scl), osteocalcin (OC), and Dkk1were determined by QRT-PCR as described in the Methods. B) Expression ofWnt7b in a mouse long was examined using in situ hybridization. Wnt7b(dark stain) is primary detected in osteoblasts. Nuclei arecounterstained in green.

FIG. 5. Effect of IIIC3 and IIC8 on the binding of sclerostin-AP toLRP5.

A) The inhibition of bone formation was inversely related to the amountof IIIC3 present. The greater the amount of IIIC3 added, the lower thepercentage of the inhibition of bone formation. B) The greater theamount of IIC8 added, the lower the percentage of the inhibition of boneformation.

1. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 2. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with at least one domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 3. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with the first and second domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 4. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 5. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with at least one domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 6. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with the first and second domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 7. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 8. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to at least one domain on the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 9. A method for promoting bone formation or bone remodeling in a patient in need thereof comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to the first and second domain on the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 10. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein said compound or said fragment of a compound comprises a small molecule, protein, peptide, polypeptide, cyclic molecule, heterocyclic organic molecule, nucleic acid, lipid, charged lipid, polar lipid, non-polar lipid, sugar, glycoprotein, glycolipid, lipoprotein or chemical.
 11. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein said compound or said fragment of a compound is an antibody or a fragment of an antibody.
 12. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 13. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with at least one domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 14. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to the site on sclerostin where sclerostin interacts with the first and second domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 15. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 16. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with at least one domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 17. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds to at least one site on sclerostin where sclerostin interacts with the first and second domain of the LRP receptor to disrupt the interaction of said sclerostin with said receptor.
 18. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 19. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to at least one domain on the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 20. A therapeutic composition for promoting bone formation or bone remodeling comprising at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, wherein said compound or fragment binds: a. to at least one site on sclerostin where sclerostin interacts with the LRP receptor; and b. to the first and second domain on the LRP receptor, thereby disrupting the interaction of sclerostin with said receptor.
 21. The composition of claim 12, 13, 14, 15, 16, 17, 18, 19 or 20 wherein said compound or said fragment of a compound comprises a small molecule, protein, peptide, polypeptide, cyclic molecule, heterocyclic organic molecule, nucleic acid, lipid, charged lipid, polar lipid, non-polar lipid, sugar, glycoprotein, glycolipid, lipoprotein or chemical.
 22. The composition of claim 12, 13, 14, 15, 16, 17, 18, 19 or 20 wherein said compound or said fragment of a compound is an antibody or a fragment of an antibody. 